Project Summary A critical factor in defining mitochondrial dysfunction during normal aging and age-associated disease is the maintenance of mitochondrial protein integrity (also referred to as protein homeostasis or proteostasis). Mitochondrial proteostasis is primarily regulated by ATP-dependent quality control proteases, including the soluble proteases, LON and CLPXP, and the membrane associated proteases, YME1L and AFG3L2. These quality control proteases regulate all aspects of mitochondrial function including energy metabolism, lipid synthesis, and apoptotic signaling. Mitochondrial quality control proteases adopt a similar ?stacked ring? organization, wherein evolutionarily similar AAA+ ATPase domains undergo ATP-dependent rearrangements to translocate protein substrates through a central pore to a proteolytic chamber for cleavage. Despite the evident structural and mechanistic similarities, mitochondrial proteases exhibit distinct proteolytic activities that allow them to regulate specific mitochondrial function. A consequence of the distinct functions of these proteases is that genetic or age-related alterations in the activity of specific proteases distinctly influences mitochondrial and cellular physiology in the context of organismal aging. Thus, an important question is ?How do these mitochondrial quality control proteases use a similar architecture to differentially influence specific aspects of mitochondrial biology?? Here, we hypothesize that unique structural features in each of these proteases have been incorporated into a generally conserved mechanism of ATP-dependent protease activity, endowing mitochondrial quality control proteases the capacity for unique biologic function. We solved the first near atomic resolution cryo-electron microscopy (cryo-EM) structure of the catalytic core of the yeast YME1L homolog YME1 bound to nucleotides and a peptide substrate, revealing the molecular mechanism responsible for ATP-dependent substrate engagement and translocation into the proteolytic chamber. We are now defining similar substrate-bound structures for the soluble AAA+ proteases LON and CLPXP using an analogous cryo-EM approach. Furthermore, we are establishing a novel unnatural amino acid platform to isolate the full-length membrane integrated (IM) proteases YME1L and AFG3L2 for structure determination by cryo-EM. By comparing structures of these evolutionarily related proteases, we are identifying the shared mechanisms that drive substrate translocation, as well as the unique structural features critical for their specific protease activities. These differences will reveal the molecular mechanisms by which aging or mutation may influence the activity of proteases, and identify new opportunities to therapeutically influence mitochondrial proteolytic activity to prevent aging- or disease-associated mitochondrial dysfunction by targeting specific aspects of protease structure.